Firefly and Renilla luciferases are widely used as reporter genes for studying gene regulation and function, and for pharmaceutical screening. The PromoKine ready-to-use Luciferase Reporter Assay Kits provide complete systems for rapid, convenient, and quantitative detection of Firefly and Renilla luciferase activity in cultured eukaryotic cells, e.g., cells transfected with a plasmid carrying the Firefly and Renilla luciferase reporter genes. They are optimized to provide excellent sensitivity as well as consistent light output when working with multiple samples.

Renilla luciferase is often used in conjunction with Firefly luciferase as a normalizing transfection control or for multiplex transcriptional reporter assays. Here, our improved Luciferase Reporter Assay Kit III (Firefly & Renilla Single-tube Assay) provides optimal convenience and highest sensitivity.
Both, Firefly luciferase and Renilla luciferase show maximum light output only when substrates and co-factors are present in large quantities. When assayed under these conditions, light emitted from the reaction is directly proportional to the number of luciferase enzyme molecules. The light emission from the reaction can be recorded using a luminometer.

Transfection rates can directly be measured by (co-)transfecting a GFP-expressing plasmid. The percentage of cells that have taken up and expressed the foreign nucleic acid can be visualized with a fluorescence microscope (by green autofluorescence).
Another way of quantification is to transfect luciferase or beta-galactosidase expressing reporter vectors and to determine the activity of these enzymes as a measure of successful transfection. Several commercial cell based reporter assays are available for this purpose (Firefly and Renilla Luciferase Reporter Assay Kits; GFP reporter Assay Kit; beta-Galactosidase Activity Assay Kit; all available from PromoKine).

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PromoKine’ s Renilla and Firefly Assays (Luciferase Reporter Assay Kits I, II, and III) are flash luminescence assays. The approximate half-lives are 1-2 minutes for Renilla and 10 minutes for Firefly. The Luciferase Reporter HTS Kit has a half-life of about 3-5 hrs. However, the signal is about 1/10 of the flash luminescence assays.

No. PromoKine’ s Renilla and Firefly Assay Kits (PK-CA707-30003; PK-CA707-30004; PK-CA707-30081) are based on flash luminescence. Samples should be treated and read individually. Alternatively, you may purchase the Firefly Luciferase Reporter HTS Kit (PK-CA707-30028). This Assay Kit is a proprietary mixture of substances that modify the enzymatic reaction to produce a long lasting signal (steady glow) by preventing the formation of adenyl-oxyluciferin at the enzyme surface.

Firefly luciferase is a 62 kDa protein isolated from beetles (Photinus pyralis), while Renilla luciferase is a 36 kDa protein from sea pansy (Renilla reniformis).
These enzymes differ in their substrate and cofactor requirements. Firefly luciferase uses luciferin in the presence of oxygen, ATP, and magnesium to produce light, while Renilla luciferase requires only coelenterazine and oxygen.
Firefly luciferase produces a greenish yellow light in the 550–570 nm range. Renilla luciferase produces a blue light of 480 nm.
These enzymes can be used in dual-reporter assays due to their differences in substrate requirements and light output.

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Renilla is often used as an internal control for transfection efficiency while the Firefly luciferase vector is used for reporter activity. They also can be reversed, with Renilla as the reporter and Firefly as the internal control.
PromoKine’s Luciferase Reporter Assay Kit III (PK-CA707-30081) allows measurement of Firefly and Renilla luciferase activity in the same sample with high sensitivity and linearity.

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Luminescence values are relative and depend on the experimental system. For example, luminometers from two different manufacturers each may have 6-log capability, but one may measure a range of 1 to 1,000,000 relative light units (RLU) while the other measures 0.01 to 10,000 RLU. Both instruments can measure a dynamic range of 6-log units, but they read out different arbitrary units.

Assay sensitivity is dependent on many factors including the promoter strength of each vector, the transfection efficiency of each vector, and the cell type used. In general, Firefly luciferase is more widely used because it has a longer half-life and better stability than Renilla luciferase.

The half life of our Renilla luciferase substrate is approximately 2 min. Although it is possible to do readings manually, I would recommend a luminometer with injectors especially if you expect small differences between samples. This removes any variability from the manual readings as it is difficult to consistently add and read the samples in a timely fashion. However, we routinely do manual readings as well and it is possible to do them this way.

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